The overall goal of this proposal is to understand each step in the process of sperm-egg plasma membrane adhesion and fusion. The first step is the contact between the tip of the acrosome-reacted sperm (the inner acrosomal membrane, IAM) and the egg plasma membrane. The second step is the adhesion between these sperm surface equatorial/posterior head region and the egg plasm membrane. Additional steps may follow adhesion and lead to membrane fusion. Previous work implicated two sperm surface proteins, fertilin and cyritestin, as acting in the equatorial/posterior head region binding step. Mutant (knockout) mice lacking fertilin were produced. The homologous, mutant males were infertile. Aim 1 proposes to use these mice to define the precise role of fertilin in gamete membrane binding, fusion and egg activation. A cyritestin gene knockout is also being generated. Aim 2 proposes to use male mice lacking cyritestin to determine cyritestin function in gamete membrane binding, fusion and egg activation. Aim 3 focuses on the first step in gamete membrane interaction, i.e. contact of the sperm IAM with the egg surface. It will be tested if this contact promotes later adhesion steps and/or the phagocytosis-like process that takes the IAM into the egg cytoplasm. Aim 4 proposes to study steps that may occur after sperm bind, but before they fuse. It will be tested if there is a metalloprotease or a surface ATP receptor that must act before fusion occurs. Aim 5 addresses the question of what molecules on the egg plasma membrane participate in interactions with the sperm that lead to fusion. It will determine if the egg surface integrin, alpha-6-beta-1, an adhesion partner for either fertilin or cyritestin. It will also propose to us a unique approach with a tissue culture cell line that fuses with mouse sperm to identify another egg protein that promotes membrane fusion. Understanding the molecular basis of the gamete fusion process may allow new developments in assisted reproduction. Furthermore, the identified, functional gamete proteins are potential targets for novel contraceptive methods based on pharmacological and immunological approaches.